Apoptosis plays important roles in the developing nervous system and in the maintenance of homeostasis in adult brain. Aberrations in the regulation of apoptosis contribute to pathogenesis of neurological diseases, including hereditary sensory and autonomic neuropathy, ALS, Huntington's Disease, Parkinson's Disease, and Alzheimer's Disease. One major mechanism to control cell death is by transcriptional regulation of neurotrophin receptors and members in the Bcl-2 family. For instance, p53 induces neuronal apoptosis by up-regulating Bax and inhibiting Bcl-xL expression. In contrast, homeodomain transcription factor Brn3a antagonizes p53 and promotes the expression of TrkA and Bcl-xL. While these results suggest that p53 and Brn3a may control the delicate balance of neuronal death and survival, it remains unclear if these two molecules interact directly or if they are regulated through a common signaling mechanism. Our data favor the latter model and indicate that homeodomain interacting protein kinase 2 (HIPK2) induces neuronal apoptosis by exerting opposing effects on p53 and Brn3a. Expression of HIPK2 in sensory neurons activates apoptosis through up-regulation of p53 target gene Bax and suppression of Brn3a targets TrkA and Bcl-xL. Consistent with these data, HIPK2-induced apoptosis can be blocked by Brn3a, Bcl-xL and Bcl-w, and is much attenuated in neurons lacking p53 or Bax. Interestingly, NGF deprivation or blockade of investigator-3 kinase results in an accumulation of HIPK2 in the nucleus of sensory neuron, suggesting that subcellular localization of HIPK2 may be regulated by neurotrophin signaling pathways. To investigate the in vivo function of HIPK2, we have generated HIPK2 null mutants and our preliminary data indicate that neurons lacking HIPK2 survive better in the absence of NGF. Taken together, these results lead to the hypothesis that HIPK2 is a key component that regulates neuronal death during development and in stress-induced pathological conditions. To test this hypothesis further, we propose to characterize the role of HIPK2 in several experimental paradigms of cell death using HIPK2-/- mutants (Aim 1). We also propose to investigate the upstream regulators of HIPK2-mediated cell death pathway and the molecular mechanisms that regulate cytoplasmic-nuclear transport of HIPK2, especially the roles of investigator-3 kinase-Akt and JNK pathways (Aim 2). Finally, we will study the mechanisms of HIPK2-induced, p53-dependent regulation of Bax and related pro-apoptotic genes. We will also determine if HIPK2-mediated activation of additional targets, such as p53-related molecule p73, contributes to neuronal apoptosis. Our long-term goal is to elucidate HIPK2-mediated signaling pathways in neuronal apoptosis. Discoveries from this project will contribute to future design of HIPK2 inhibitors that can promote neuronal survival in injury and degenerative conditions.